The current challenges facing oncology practitioners and researchers include the development of specific treatments and efficient tumour detection methods. Genetically modified, pathogenic and non-pathogenic bacteria have begun to emerge as potential biological agents with natural tumour specificity. Several bacterial species or genera (e.g. Escherichia coli, Bifidobacterium, attenuated Salmonella typhimurium, Clostridium, Vibrio cholera, Listeria monocytogenes) have been demonstrated to localise to and replicate in tumour tissue when intravenously administered in rodent models. Achieved through anaerobic bacterial targeting, specific localisation of species, such as members of the Bifidobacterium genus, in hypoxic regions of tumours after intravenous administration has been reported.
The term bacterial translocation refers to trafficking of viable bacteria from the Gastro-Intestinal Tract (GIT) to extraintestinal sites. This phenomenon is well studied in bacterial sepsis linked with various conditions [14,15]. The three primary mechanisms promoting bacterial translocation in animal models are identified as: (a) disruption of the ecologic GI equilibrium to allow intestinal bacterial overgrowth, (b) increased permeability of the intestinal mucosal barrier, and (c) deficiencies in host immune defences. These mechanisms can act in concert to synergistically promote the systemic spread of indigenous translocating bacteria to cause lethal sepsis. In animal models in which the intestinal barrier is not physically damaged, pathogenic bacteria may translocate by an intracellular route through the epithelial cells lining the intestines and then travel via the lymph to the mesenteric lymph node (MLN). In animal models exhibiting damage to the mucosal epithelium, indigenous bacteria translocate intercellularly between the epithelial cells to directly access the blood. Indigenous gastro-intestinal (GI) bacteria have been cultured directly from the MLN of various types of patients. Thus, evidence is accumulating that translocation of indigenous bacteria from the GI tract is an important early step in the pathogenesis of opportunistic infections originating from the GI tract. Sampling from humans has indicated that translocation may be a phenomenon that occurs in healthy individuals and may be a normal physiologic event without deleterious consequences [48].
Bifidobacteria are a native, harmless resident of the human gut, and certain bifidobacterial strains have been shown to have health-promoting or probiotic benefits. A number of bifidobacterial strains that harbour plasmids expressing therapeutic agents, such as endostatin or prodrug activating enzymes, have been shown to induce regression in rodent tumour models when administered intravenously [33]. Live imaging of mice has shown intravenously administered lux tagged invasive pathogenic bacteria replicating locally in tumours. No imaging of bifidobacteria in tumours has been reported.
To date, bacterial localisation to tumours has been described only with intravenous administration. Fu et al previously described use of B. longum to deliver a therapeutic peptide to the GIT [33]. B. longum expressing endostatin was administered orally to athymic mice, and the authors reported that subsequent gut absorption of the therapeutic peptide resulted in slowing in the growth of subcutaneous (s.c.) liver tumours. Translocation of bacteria from the gut to tumours was not reported. Several studies have investigated the ability of bifidobacterial colonisation of the GIT to inhibit translocation of pathogens [16]. A study from 1985 demonstrated B. longum colonisation of organs post feeding [47]. The authors reported very low bacterial counts in the kidney and liver however. In addition the authors did not report bacterial translocation to tumour cells.
Despite the teachings of the prior art, the current inventors have surprisingly demonstrated translocation of a non-pathogenic species of bacteria following oral administration, utilising a lux luminescence-based tagging system in Bifidobacteria breve in mice, with subsequent growth specifically in tumours. B. breve UCC2003 carrying the plasmid pLuxMC3 [18] was orally administered to athymic MF1 nu/nu mice bearing subcutaneous (s.c.) B16-F10 murine melanoma tumours (FIG. 1a). By transfection with plasmids that are suitable for bacterial replication expression of heterologous genes, such bacteria can home to tumours, replicate within them and locally express therapeutic proteins. It would not have previously been expected by those skilled in the art that non-pathogenic viable species of bacteria would translocate to tumours in hosts.
The current invention has the added advantage in that it necessitates oral administration, which is relatively painless and offers greater flexibility for use in a range of clinical situations and is of particular benefit for paediatric patients. The administration of therapeutic agents intravenously, as described previously, is painful and causes patient discomfort.